Discovery of a new Hepatozoon species namely Hepatozoon viperoi sp. nov. in nose-horned vipers in Türkiye

Although Hepatozoon spp. remains the most prevalent intracellular protozoa infecting snakes, it was reported only in a few snake species of the Colubridae family in Türkiye. Moreover, studies on these hemoparasites are not available in venomous nose-horned vipers from Türkiye. In this study, we investigated Hepatozoon spp. in three individual Vipera ammodytes using morphological and molecular methods. Our results were positive for intraerythrocytic Hepatozoon spp. gamonts in all three snakes, exhibiting low parasitemia. The microscopic findings were further confirmed through molecular data. A genus-specific PCR assay targeting the 18S rRNA gene region of Hepatozoon spp., was performed using HemoF/HemoR and Hep300/Hep900 primers. The obtained sequences were concatenated and used in phylogenetic analyses in comparison with different Hepatozoon species. Although our (OP377741) isolate was separated into a different branch, it was clustered with the isolates of H. massardi (KC342526), H. cevapii (KC342525), and H. annulatum (ON262426) from Brazilian snakes. Moreover, gene similarity and pair-wise distance between our isolate and other Hepatozoon species infecting snakes were found to be 89.30–98.63% and 0.009–0.077, respectively. Hence, we reported a new species of Hepatozoon, namely Hepatozoon viperoi sp. nov. infecting V. ammodytes. Since the literature does not indicate the existence of such a Hepatozoon species in V. ammodytes in different countries, our data may contribute to the expanding knowledge of Hepatozoon species in snakes, providing new insights into the biodiversity of the haemogregarine protozoan parasite.

Furthermore, dehydration, lethargy, open-mouth breathing, and weight loss may be encountered in immunecompromised and aberrant hosts 7 .
More than 340 Hepatozoon species cause diseases in animals having veterinary importance, including domestic and wild animals 8 . Previously, new Hepatozoon species or isolates have been detected in different countries in various snake species through advanced molecular diagnostic techniques [9][10][11][12][13] . However, many of them were related to the Hepatozoon species in colubrid snakes, with restricted information available on viper snakes. Studies are particularly scarce on the snakes belonging to the Vipera genus of the Viperidae family. Also, some studies have not shown the expected results 14,15 . Although some Hepatozoon species were detected in various animals in Türkiye 6 , one study in particular reported Hepatozoon infections in colubrid snakes 15 . However, the investigation of hemogregarine protozoans has not been done at the microscopic or molecular level in nosehorned vipers i.e., Vipera ammodytes in Türkiye.
Thus, the main objective of this study was to detect the presence of Hepatozoon species in nose-horned vipers and determine their phylogenetic relationships with various species or isolates of other snakes and reptiles using morphological examination and molecular techniques.

Material and methods
Study animals. The study consisted of three female nose-horned vipers (Vipera ammodytes Linnaeus, 1758) captured in 2022 from the Göztepe locality (41° 35′ 3.71″ N, 27° 47′ 48.70″ E) of the Vize district of Kırklareli province, Türkiye. Nose-horned vipers are venomous species and generally live in stony and rocky areas. For this study, the vipers were captured using a snake grab stick. Figure 1 shows the locality from where these nosehorned vipers were captured.
Blood samples and microscopic examination. The nose-horned vipers were firstly restrained to collect blood. Then, approximately 2 mL of blood was collected through the post-orbital sinus in heparinized hematocrit tubes 17,18 . Next, the blood samples were transferred to a tube containing ethylenediaminetetraacetic acid (EDTA), and thin blood smears were prepared from each blood sample. The samples were then air dried, fixed in absolute methanol for 5 min, and stained for 20 min using Wright's staining method 19 . After smear preparation, the remaining blood samples were stored at − 20 °C for further molecular analysis while the captured horned vipers were released back to their habitats. All smears were examined under a light microscope (Olympus CX 31) at 100× magnification. Relevant literature was used to identify the gamont stages of intraerythrocytic parasites 3 .  PCR amplification and pathogen detection. We performed two PCR assays targeting two different regions of the 18S rRNA gene of apicomplexan parasites by using two primer sets. The HemoF/HemoR primers were used to amplify the 900 bp region while Hep300/Hep900 was used to amplify 600 bp of the 18S rRNA gene region of Hepatozoon spp. 20,21 . PCR was performed by making minor modifications to the PCR thermal cycles. Sanger sequencing and phylogenetic analysis. PCR products were sequenced in both directions by a commercial company. The obtained sequence chromatograms (forward and reverse sequences) were assembled, and each primer sequence (HepF300/900 and HemoF/R) was edited out to obtain a partial 18S rRNA consensus sequence. These sequences were concatenated (~ 1200 bp) using the Geneious version 7.1.3 (Bomatters, "http:// www. genei ous. comww") 22 . Also, the sequences belonging to the haemogregarine group in Genbank were aligned using Geneious version 7.  27 . Phylogenetic analysis was inferred using PhyML with 1000 replicate bootstraps (> 50%) 28 . The BI analysis was carried out using MrBayes implemented from the computational resource CIPRES 29 , the best BIC score was the General Time Reversible model (GTR + I + Γ) 30 . The Markov chain Monte Carlo (MCMC) algorithm was run for 10,000,000 generations, sampling one tree every 1000 generations. On the burn-in, the first 25% of generations were discarded, and the consensus trees were estimated using the remaining trees. Bayesian posterior probabilities (BPP) cut off was considered > 50%. The aligned sequences of haemogregarine species infecting snakes were compared using a pair-wise distance (p-distance) matrix.

Accession number status.
We submitted the sequences obtained from two different primer sets (HemoR/F, Hep300/900) targeting different regions of the 18S rRNA gene region to the GenBank, National Center for Biotechnology Information with the following accession numbers: ON619569/OM866261 (VA1), OM866262/OM866263 (VA2) [HemoR/HemoR], and ON673853/ON673854 (VA1), ON629811/ON629812 (VA2) [Hep300/Hep900]. However, since these sequences represented a short part of the target gene region, they were not included in the phylogenetic analyses. Instead, contig sequences amplified using both primer pairs were concatenated to obtain a sequence representing a longer part (~ 1200 bp) of the 18S rRNA gene (OP377741), which was then used in phylogenetic analyses (https:// www. ncbi. nlm. nih. gov/ nucco re/ OP377 741.1/). ). Data were evaluated after controlling for normal distribution prerequisites (Kolmogorov-Smirnov test). Since the differences between the two independent groups did not meet the parametric test prerequisites, the Mann-Whitney U test was used. Variables were expressed as median (min/max) values. The values of p < 0.05, p < 0.01, and p < 0.001 were accepted as the significance levels of the tests.

Results
We found that all three nose-horned vipers (V. ammodytes) in the Thrace Region of Türkiye were infected with the Hepatozoon species. The infected vipers were adult females with low parasitemia. We observed mature gamonts of Hepatozoon in the erythrocytes of the hosts. Since we did not perform histopathological examinations, we could not evaluate other developmental stages of the parasite. The molecular and phylogenetic analysis verified the species as an undescribed Hepatozoon spp. in V. ammodytes. Site of infection: erythrocytes (blood). Vector: unknown. Etymology: considering the genus name of the infected host (Vipera ammodytes), the protozoan was named Hepatozoon viperoi sp. nov. This is the first Hepatozoon species reported in the nose-horned viper belonging to the Vipera genus.

Species description.
Parasitemia: 0.1-0.4%. Deposited materials: blood smears and DNA samples from V. ammodytes. These materials are deposited in Selcuk University, Faculty of Veterinary Medicine, Department of Veterinary Parasitology.
Gene sequence: the 18S rRNA concatenated gene sequence obtained from the blood of V. ammodytes was submitted to GenBank under the accession number (OP377741).

Microscopy.
We found that the nose-horned viper V. ammodytes was infected with a Hepatozoon species.
In this study, gamonts of Hepatozoon spp. were detected microscopically in thin blood smears of all captured horned vipers. Figure 2 shows different microscopic images of gamonts in each snake, separately. Although the parasitemia level was quite low (< 0.1%) in VA1 and VA3 samples, it was slightly higher in VA2 (0.4%) sample. Morphological and morphometric analysis of gamonts. Gamonts of the Hepatozoon viperoi sp. nov.
were detected only in the erythrocytes of V. ammodytes, with no other developmental stages being observed in the thin blood smears. Gamonts were enclosed in a thin parasitophorous vacuole. They were elongated and had one end more tapered while the cytoplasm was stained whitish pink. Sausage-shaped gamonts were found near the nucleus of the infected erythrocytes, exhibiting a uniform cytoplasm. Furthermore, gamonts displaced the nucleus of the host erythrocytes. Table 2 presents the morphometric parameters including the length and width of the erythrocytes and the length and width of the nucleus of the gamonts. The pinkish-purple gamont nuclei were centrally located and were mostly quadrangular in shape. The length, width, and area of the gamonts (mean ± standard deviation) were 14.05 ± 0.44 µm, 5.83 ± 0.42 µm (n = 15), and 64.96 ± 6.64 µm 2 , respectively. The length, width, and area of the nucleus of the parasite were 6.35 ± 0.65 µm, 5.20 ± 0.47 µm, and 22.91 ± 3.25 µm 2 (n = 20), respectively. The nucleus of the infected erythrocyte had an elongated appearance and was compressed between the gamont and the membrane of the erythrocyte. The infected host erythrocytes showed significant hypertrophy (P < 0.05) along with higher mean length (P < 0.001) and width (P < 0.05). Although the areas of infected erythrocytes were significantly higher than the areas of non-infected erythrocytes (P < 0.001), the area of their nuclei remained smaller (P < 0.05). The length, width, and area of the infected erythrocytes were 18.31 ± 0.71 µm, 11.91 ± 0.88 µm (n = 15), and 165.07 ± 11.17 µm 2 (n = 20), respectively, while the length, width, and area of the uninfected erythrocytes were 15.60 ± 0.69 µm, 11.22 ± 0.66 µm (n = 15), and 144.83 ± 17.11 µm 2 (n = 20), respectively. Table 2 indicates the detailed measurement values of the morphometric parameters.

Remarks.
Although the literature review reported on the presence of Hepatozoon spp. in some snakes of Türkiye, no Hepatozoon species have been formally described (using morphological or a combined approach of morphology and molecular) in snakes so far. Tome et al. detected Hepatozoon spp. at the molecular level in some colubrid snakes, including Dolichophis caspius, Elaphe sauromates, and Natrix tessellata, however, the isolates were not identified at the species level 15 . This said, no morphological or morphometric data on Hepatozoon species are available in snakes in Türkiye. The morphological and morphometric characteristics of Hepatozoon viperoi sp. nov. determined in our study can be compared to Hepatozoon species found in some Brazilian snakes, which may show a phylogenetic relationship. To date, although no studies have reported the occurrence of Hepatozoon species in V. ammodytes, some new species including H. cuestensis, H. cevapii, and H. massardi have been described in rattlesnakes (Crotalus durissus terrificus). These rattlesnakes belong to the genus Crotalus, subfamily Crotalinae, and family Viperidae. Among these, H. cevapii and H. massardi showed phylogenetic proximity to H. viperoi sp. nov. described in our study. Therefore, we used these two species for the morphological and morphometric comparison. Moreover, we included a recently described new species H. annulatum, found in a The length, width, and area of the H. cevapii gamonts found in the colubrid snake species Oxyrhopus rhombifer in Brazil were 14.81 ± 0.99 µm, 5.02 ± 0.76 µm, and 64.38 ± 4.70 µm 2 , respectively, while the length, width, and area of the parasites' nucleus were 4.35 ± 0.28 µm, 3.99 ± 0.98 µm, and 14.01 ± 1.87 µm 2 (n = 30), respectively 13 . The area of the H. cevapii gamonts was similar to that of the H. viperoi sp. nov., with minor differences in other morphometric measurements. However, significant morphometric differences were observed between the gamonts' nuclei. The values of the nuclei length, width, and area of H. viperoi sp. nov. gamonts were higher than that of H. cevapii.
The length, width, and area of the H. massardi gamonts found in Crotalus durissus terrificus species in Brazil were 17.4 ± 0.7 µm, 3.0 ± 0.3 µm, and 38.9 ± 3.7 µm 2 , respectively, while the length, width, and the area of the parasites' nucleus were 4.7 ± 0.3 µm, 2.3 ± 0.2 µm, and 9.1 ± 0.8 µm 2 , respectively 9 . Although the gamont length of H. viperoi sp. nov. was shorter than that of H. massardi, the other morphometric measurements including the gamont width and area and the nucleus length, width, and area were higher in the gamonts of H. viperoi sp. nov.
The length, width, and area of H. annulatum gamonts described in Leptodeira annulata, a colubrid snake species in Brazil, were 14.25 ± 0.54 µm, 5.34 ± 0.26 µm, and 64.32 ± 5.90 µm 2 , respectively, while the length, width, and the area of the parasites' nucleus were 3.91 ± 0.63 µm, 4.13 ± 0.29 µm, and 16.95 ± 2.01 µm 2 , respectively 13 . The morphometric measurements of the H. viperoi sp. nov. including the gamont's length, width, and area were found to be very similar to that of H. annulatum. However, the measurements of the nucleus of the gamont showed significant differences between both species.
Hepatozoon. viperoi sp. nov. detected in our study showed phylogenetic similarity with H. cevapii, H. massardi, and H. annulatum species found in various Brazilian snakes. However, these three Hepatozoon species have not been detected in Türkiye yet. The absence of morphological-morphometric studies of Hepatozoon spp. in the snakes of Türkiye led us to compare our findings with those found in the snakes of other countries. Although morphological examination plays a significant role in the differential diagnosis, it is still difficult to predict the Hepatozoon species without morphometric measurements, which requires further molecular confirmation. Molecular data, sequencing, and phylogenetic analysis. Genomic DNAs were amplified using two sets of primers (HemoF/HemoR, Hep300/Hep900) targeting different regions of the 18S rRNA gene of Hepatozoon. Amplified products were sent for sequencing, and the obtained sequences were submitted to the GenBank database of the National Center for Biotechnology Information. The contig sequences obtained using the pair of primers HemoF and HemoR were further aligned and compared using the Geneious version 7.1.3 (Bomatters, www. genei ous. comww) 22 . Since these sequences were 100% similar, another pair of primers (HepF300/900) was used to amplify another part of the 18S rRNA gene, which was then concatenated with the sequence amplified using HemoF/HemoR resulting in a ~ 1200 bp sequence (OP377741).
Both Bayesian Inference (BI) and the Maximum Likelihood (ML) phylogenetic analyses resulted in identical tree topologies (Fig. 3). The phylogenetic analyses included isolates of adeleorinid parasites (Haemogregarinidae, Hepatozoidae, Karyolysidae, and Dactylosomatidae) available in GenBank. Hepatozoon spp. belong to polyphyletic groups that form two separate clades according to their vertebrate host species. Hepatozoon spp. isolated from large mammals form a sister clade with the Karyolysus genus while the isolates obtained in our study clustered with the sequences of the reptile and anuran hosts belonging to a large Hepatozoidae clade. Although our isolate clustered with the isolates obtained from Brazilian snakes, including Hepatozoon massardi O´Dwyer, Moço, Paduan, Spenassatto, Silva and Ribolla 2013 (KC342526), Hepatozoon cevapii O´Dwyer, Moço, Paduan, Spenassatto, Silva and Ribolla 2013 (KC342525/ON236891) and Hepatozoon annulatum Úngari, Netherlands, Silva and O´Dwyer 2022 (ON262426), it was clustered on a different branch. The gene similarity and pair-wise distance are summarized in Table 3.

Discussion
The snake species Vipera ammodytes (Linnaeus, 1758) is characterized by its prominent fleshy horn at the tip of the snout. As one of the most venomous vipers in Eurasia, V. ammodytes are widely distributed between Austria and Italy, and across the Balkan Peninsula to Türkiye and Caucasus 31,32 . These vipers live in mesophytic and xerophytic forests, dry cliffs, mosaic meadows, screes, scrubs, and even on artificial stone walls. They commonly prey on lizards and small mammals by subduing them with their venom 32,33 . Telford stated that the infection of the snakes with Hepatozoon species may be based on the predation of infected vertebrate hosts or the ingestion of the invertebrate vectors 3 . Therefore, in this study, we investigated the existence of Hepatozoon spp. in vipers. The microscopic findings revealed the intraerythrocytic sausage-shaped gamonts. However, since microscopy was inadequate for species identification, we further diagnosed it molecularly 34 .
A wide variety of pathogens can infect reptiles, with some groups of parasites receiving little attention. As a result, their biodiversity awaits clarification. Systematics of parasites commonly rely on some features, including the morphological and lifecycle characteristics of parasites, some of which are difficult to evaluate. However, molecular techniques have eliminated these difficulties, in turn, increasing the knowledge of their biology, biodiversity, and phylogeny [34][35][36] 14 . Nasiri et al. conducted a study on parasites in Iranian snakes and investigated three species from the Vipera genus but found no blood parasites in the zigzag mountain vipers (Vipera albicornuta) and Transcaucasian meadow vipers (Vipera ursinii eriwanensis) 38 . Although they observed intraerythrocytic gametocytes of hemoparasites in West-Asian blunt-nosed vipers (Vipera lebetina obtusa), they did not perform any molecular confirmation at the species or genus level. The data remains insufficient on the molecular or microscopic prevalence of Hepatozoon spp. in vipers belonging to the genus Vipera, however, these parasites were detected in several viper species belonging to other genera of the Viperidae family 9,14,15,[37][38][39][40][41][42]   .95%, PD: 0.072-0.077). Clustering of our concatenated sequence with the sequences from the reptile and anuran host revealed a relationship between the similarities in the detected nucleotides and the feeding or hunting patterns of nose-horned vipers. Tome et al. stated that the genetic lineages infecting the prey may also parasitize snakes 4 . Our study findings also supported this view, however, further studies encompassing a wider sample of vipers may be required to establish clearer genetic proximities between snakes and prey.
Vipera ammodytes are among the strictly protected fauna species mentioned in Annex II of the Bern Convention on the Conservation of European Wildlife and Natural Habitats 45 . Also, recently, it has been listed in the Least Concern (LC) category according to the International Union for Conservation of Nature (IUCN) criteria 46 . Detailed investigations are necessary on whether Hepatozoon species or other infectious disease agents could affect various physiological activities of vipers along with exploring the extent of the parasitic effect on them since vipers hold an important place in terms of the biodiversity of Türkiye's herpetofauna. Because diseases occurring in natural populations can adversely affect their chances of survival, mainly the reproductive functioning of living beings, it is crucial to take necessary protective actions. One of the main constraints in studying parasites remains the limited comparative genetic data of different host species, especially, wild populations belonging to various geographical areas. Our study reported a new Hepatozoon species in nose-horned viper V. ammodytes, which has not been studied previously. Hence, this study can contribute to the lack of literature in this regard.
Wildlife species include unique clues allowing early detection of changes in the ecosystem, including habitat fragmentation, environmental pollution, and the presence of pathogens. Wildlife has been frequently considered as sentinels for pathogen emergence and persistence [47][48][49][50] . Therefore, studies regarding the haemoparasites of snakes, a significant part of wildlife, becomes important. In conclusion, we used morphological and molecular techniques to describe a new Hepatozoon species (Hepatozoon viperoi sp. nov.) infecting V. ammodytes found in the Thrace region of Türkiye. To the best of our knowledge, this is the first study reporting a haemoprotozoan parasite in nose-horned viper in Türkiye. The identification of a new Hepatozoon species may increase the knowledge of parasitic fauna infecting nose-horned viper and also their biodiversity, which has been poorly studied. Therefore, we believe that this study may help identify unknown threat factors, which in turn, may be required for generating conservation strategies for nose-horned viper.

Data availability
All data generated or analyzed during the current study are included in this article. The concatenated sequence has been deposited in the NCBI GenBank database under the accession number OP377741 (https:// www. ncbi. nlm. nih. gov/ nucco re/ OP377 741.1/).